PROJECT SUMMARY/ABSTRACT Although 3 major classes of systemic antifungal agents are clinically available, each is characterized by important limitations. Azole antifungals, although usually well-tolerated, are limited by resistance within Candida albicans biofilms and in several important non-albicans Candida species. Amphotericin, including lipid-formulations, while broadly effective against many fungal pathogens, is associated with substantial toxicities. The echinocandins, while highly effective against most Candida species, can be ineffective in the presence of point mutations in the FKS1 gene which encodes the glucan synthase drug target. We propose a high throughput screening project to use small molecule libraries to probe a key membrane protein, the ABC transporter Cdr1p, which is associated with antifungal drug resistance in the model opportunistic fungal pathogen C. albicans and other Candida species. CDR1, which encodes an ABC-type efflux pump, is overexpressed in C. albicans laboratory and clinical isolates that are phenotypically resistant to fluconazole and other azole antifungal drugs. Furthermore, the level of CDR1 expression correlates with the degree of azole resistance. Genetic deletion of CDR1 leads to increased susceptibility to azoles. Cdr1p thus represents a potential drug target, which if inhibited, should lead to reduced resistance to azole antifungal drugs. To identify specific inhibitors of Cdr1p expression, we propose a cell-based high throughput, flow cytometry assay to screen a selected set of commercially available compound libraries and the Torrey Pines Institute Molecular Library to identify small molecules which modify expression of Cdr1p. This screen should identify inhibitors of Cdr1p expression, which is a novel approach to Cdr1p inhibition. We therefore used azole-resistant clinical isolates that overexpress CDR1 to construct strains bearing C- terminal GFP tags. We will screen these C. albicans strains bearing intrinsically over-expressed CDR1-GFP in a high-throughput format, as these strains are highly amenable to fluorescence-based flow cytometric analysis. Changes in fluorescence, either inhibition or increased expression, will imply modulation of the drug target. Utilizing an institutional CTSC pilot grant, we have already successfully screened these strains using this high- throughput assay against the SPECTRUM compound library (see Preliminary Studies). After identification of initial hits and confirmation of fluorescence modulation using dose-response curves, these hits will be analyzed in secondary and tertiary assays for specific (i) Cdr1p expression and transporter activity, (ii) antifungal activity in combination with fluconazole, and (iii) effects on virulence. The most promising lead compounds will be studied for structure-activity relationships and lead optimization using medicinal chemistry for maximal therapeutic activity, followed by testing in a murine treatment model in vivo.